In the assay of body fluids, especially blood serum, for cholesterol concentration, the initial step requires hydrolysis of cholesterol esters to free cholesterol.
Conventional procedures for cholesterol ester hydrolysis use a strong base (KOH, NaOH, etc.), or for reasons of simplicity and selectivity, a hydrolase enzyme (i.e., a cholesterol esterase). Handling of caustic materials may be inconvenient or undesirable and, as discussed in relation to prior publications below, while enzymatic techniques can be useful for the hydrolysis of "free" cholesterol esters, i.e., those not bound to protein, they are either ineffective or very slow when used to treat protein-bound cholesterol esters. The binding of the ester to protein apparently inhibits the action of the esterase and thus requires some means for breaking the protein-ester complex before the enzyme can act on the ester.
U.S. Pat. No. 3,869,349 issued Mar. 4, 1975 to Goodhue et al describes an improved technique for hydrolyzing serum cholesterol esters which involves the use of compositions comprising a lipase preparation which demonstrates cholesterol esterase activity and a protease. This patent contains no suggestion that a surfactant can replace the protease.
U.S. Pat. No. 3,703,591 to Bucolo et al describes the use of a combination of a lipase and a protease to achieve serum (i.e., protein-bound) triglyceride hydrolysis. No suggestion is made to use a surfactant either in combination with or as a substitute for the protease in the hydrolysis of cholesterol esters.
U.S. Pat. No. 3,759,793 to Stork et al describes the hydrolysis of serum triglycerides using a lipase from Rhizopus arrhizus which is apparently identical to that suggested by Bucolo et al, however, with no requirement for a protease. The reasons for this apparent anomaly are not clear; however, it is noted in British Pat. No. 1,395,126 of the same assignee that the Stork et al hydrolysis technique is very slow. This British Patent describes an improved method for hydrolyzing triglycerides with the aforementioned Rhizopus arrhizus lipase comprising contacting the triglyceride with the lipase in a buffer and in the presence of carboxylesterase and an alkali metal or alkaline earth metal alkyl sulfate, the alkyl radical of which contains 10 to 15 carbon atoms. The preferred alkyl sulfate is sodium dodecyl sulfate. There is no suggestion that the use of surfactant alone in the absence of carboxylesterase stimulates hydrolase activity of triglycerides.
Helenius, Ari and Simons, Kai, Biochemistry, Vol. 10, No. 13 (1971) describe a method for removing all major lipids from human plasma low-density lipoprotein comprising treatment of the human plasma with high concentrations of natural and synthetic surfactants. Lipid removal is applied for purposes of characterizing the lipid free protein moiety of human plasma low-density lipoprotein. There is no suggestion in this publication that the combination of a surfactant and a lipase would yield a useful analytical tool which would simplify the assay of serum for cholesterol content by providing a fast and accurate hydrolysis method and a stable assay composition.
U.S. Pat. No. 3,689,364 issued Sept. 5, 1972 describes an assay for lipase contained in body fluids such as blood serum using a "free" triglyceride emulsion as substrate for the lipase. It is suggested that the bile salts which stabilize the substrate emulsion of "free" triglyceride (i.e., triglycerides not bound to protein) also concurrently exert an "activating effect" on the lipase under assay when it is a pancreatic lipase. The activating effect apparently results in an increase in the hydrolytic activity of the lipase on the free triglycerides of the substrate emulsion. There is no teaching or suggestion in this patent that such bile salts exert any effect on lipase preparations when contacted with lipids bound to proteins as are found in blood serum. In particular there is no suggestion that such lipase can hydrolyze protein-bound cholesterol esters.
U.S. Pat. No. 3,898,130 to Komatsu issued Aug. 5, 1975 describes a method for hydrolyzing triglycerides comprising contacting triglyceride with a composition comprising a mixture of a microbial lipase, particularly Candida lipase (sic), a pancreatic lipase and a bile salt selected from sodium taurodeoxycholate, taurocholate, taurochenodeoxycholate and taurodehydrocholate. Both the microbial and the pancreatic lipase enzymes are critical components of the hydrolysis composition.
German Offenlegungsschrift No. 2,522,432 published Dec. 4, 1975 describes an enzymatic method for hydrolyzing cholesterol esters using a cholesterol esterase from Pseudomonas fluorescens. There is no suggestion of the use of or a need for a surfactant to achieve protein-bound cholesterol ester hydrolysis.
French Pat. No. 2,223,696 and U.S. Pat. No. 3,925,164 issued Dec. 9, 1975 describe an assay for total cholesterol in blood serum wherein cholesterol esters are hydrolyzed with an enzyme preparation from Candida rugosa, Rhizopus or Aspergillus in the presence of a surfactant. The only suggested surfactant is hydroxypolyethoxy dodecane. As will be shown in the examples below, such surfactants are not as effective as the materials described herein, possibly because of incompatibility with the cholesterol ester hydrolase.
German Offenlegungsschrift No. 2,509,156 published Sept. 23, 1975 describes an enzymatic method for the assay of total cholesterol using as the cholesterol ester hydrolyzing medium cholesterol esterase and a gallic acid or a salt of a gallic acid. The cholesterol esterase is identified as EC 3.1.1.13 which is derived from Nocardia restrictus. There is no suggestion in the foregoing publication that the synthetic surfactants described herein are useful effectors for cholesterol esterase.